Setsuko Hara

Preparation of polyunsaturated phospholipids by lipase-catalyzed transesterification

AbstractTransesterifications were investigated to determine a means for preparing polyunsaturated phospholipids simply from soy phospholipid, sardine oil, and two kinds of microbial lipases originating fromCandida cylindracea andRhizopus delemar.The optimum reaction conditions forCandida cylindracea lipase were: 4 g of sardine oil, 10 mL of water, 0.7 g of lipase, 10 mL of hexane, 48 hr of reaction time at 37°C for 3 g of soy phospholipid, for which the transesterification ratio reached approximately 45%. Recovery of phospholipid was low, because hydrolysis also occurred under these reaction conditions. However, hydrolysis could be suppressed by using glycerine instead of water, and the recovery of phospholipid increased to 47%, although the transesterification ratio was reduced to 32%. Rhizopus delemar lipase has 1,3-specificity for triglycerides, and the transesterification ratio was approximately 37% in the 1-position of phospholipid. The resulting phospholipid was rich in polyunsaturated fatty acids and linoleic acid, while the total percentage of polyunsaturated fatty acids incorporated was 18.4%. Therefore, polyunsaturated phospholipids can be prepared easily by transesterification of soy phospholipid with fish oil by means of commercial lipases.

A highly sensitive method for the micro-determination of lipid hydroperoxides by potentiometry

A modified method for peroxide value (POV) determination of lipids was developed through the application of potentiometry to conventional POV tests such as the official method of the Japan Oil Chemists’ Society (JOCS). The new method permits a simple and reliable determination of low hydroperoxide levels in the initial stages of lipid autoxidation when only very small amounts of sample are available, even when those levels are measured on less than 10 mg of lipid. Using the present method, hydroperoxide levels as low as 20 nanoequivalents (neq) were determined with reasonable precision. This method is applicable to all lipids tested including oils and fats, free fatty acids, phospholipids, glycolipids and cholesterol esters.

Enzymatic Preparation of Human Milk Fat Substitutes and Their Oxidation Stability

Human milk fat substitutes (HMFSs), rich in palmitic acid (P) at the sn-2 position of triacylglycerol (TAG), were prepared from lard via Novozym435(®)- and Lipozyme RM-IM(®)-mediated two-step reactions. First, 2-palmitoyl monoacylglycerol (2-P-MAG, 90% purity) was prepared via Novozym435(®)mediated ethanolysis of lard. Then, 2-P-MAG, oleic acid (O), linoleic acid (L), and lard were dissolved in hexane and subjected to a Lipozyme RM-IM(®)-mediated reaction for HMFS preparation. The effect of the amount of 2-P-MAG and fatty acids (O and L) in HMFS preparation were investigated. Under the optimum reaction conditions: 7 mmol of lard, 3.0 mmol of 2-P-MAG, 5.2 mmol of O, 3.5 mmol of L, 10 mL of hexane, 10 wt% of Lipozyme RM-IMR(®) (against the total weight of substrates), 550 rpm, 37°C and 6 h, a HMFS with total fatty acid composition and P content at the sn-2 position of TAG similar to that of human milk fat was prepared. In the same way, a HMFS having polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (PUFA-HMFS) was prepared. The HMFS and PUFA-HMFS prepared in this study, as well as fats extracted from commercially available powdered milk for infants (FPM) were evaluated for their oxidation stability by an auto-oxidation test. The test showed HMFS and PUFA-HMFS to possess greater oxidation stability than that the FPMs. These results indicated that the HMFS and PUFA-HMFS prepared in this study have value as potential ingredients for powdered milk.

Preparation of Chiral Triacylglycerols, sn-POO and sn-OOP, via Lipase-mediated Acidolysis Reaction

It is well known that lipases are useful tools for preparing various structured triacylglycerols (TAGs). However, the lipase-mediated preparation of chiral TAGs has never been reported. This study aimed to prepare chiral TAGs (viz., 1-palmitoyl-2,3-dioleoyl-sn-glycerol (sn-POO) or 1,2-dioleoyl-3-palmitoyl-sn-glycerol (sn-OOP)) via lipase mediated acidolysis, using triolein (TO) and palmitic acid (P) as substrates. Three commercially available lipases (viz., Lipozyme RM-IM®, Lipozyme TL-IM®, and Lipase OF®) were used. Lipozyme RM-IM® resulted in an increase 1P-2O (sn-POO + sn-OOP + 1,3-dioleoyl-2-palmitoyl-sn-glycerol) content with reaction time, which plateaued at 2~24 h (max. yield 47.1% at 4 h). The highest sn-POO/sn-OOP ratio of ca. 9 was obtained at 0.25 h, and the rate got close to 1 with reaction time (sn-POO/sn-OOP = 1.3 at 24 h). Lipozyme TL-IM® resulted in a lower 1P-2O synthesis rate than Lipozyme RM-IM®, where its highest sn-POO/sn-OOP ratio of ca. 2 was obtained at 0.25 h and did not vary much further with reaction time. In the case of Lipase OF®, its reaction rate for 1P-2O synthesis was lower than that of the other two lipases, and the highest sn-POO/sn-OOP ratio of ca. 1.4 was obtained at 0.5 h, reaching closer to 1 with a longer reaction time. Reaction solvents (viz., hexane, acetone, and benzene) also affected the 1P-2O preparation, where the highest 1P-2O content was obtained with the solvent-free system. Furthermore, the solvent-free system showed a higher reaction rate for 1P-2O synthesis than did the hexane system, with no effect on chiral specificity of the lipase for the TAG molecules. These results suggested that among three types of commercial lipase, Lipozyme RM-IM® is the most useful for the preparation of chiral TAGs by acidolysis reaction.

Effects of Triacylglycerol Molecular Species on the Oxidation Behavior of Oils Containing α-Linolenic Acid.

Two kinds of oils, pure perilla oil and a blend of perilla oil with palm oil, and their enzymatically interesterified oils having the same fatty acid compositions but with different compositions of triacylglycerol (TAG) species, were studied. In particular, the effects of TAG molecular species on the oxidation resistance of oils containing α-linolenic acid (Ln) were investigated. The content of TAG binding to three Ln molecules (3Ln-TAG) was found to be different between perilla oil (38.7%) and the interesterified oils (14.5-28.9%) , which were generated using Lipozyme RM-IM(®) (regiospecificity: sn-1, 3 positional). Oils with lower 3Ln-TAG contents were more stable to oxidation as determined by the conductometric determination method (CDM; 90°C, 20 L/h) than oils with higher 3Ln-TAG contents. This result was also supported by heating oxidation tests (180°C, 7 h) using the interesterified blended oils; the residual ratio of Ln-TAGs in the oils was found to be in the order of 3Ln-TAG<2Ln-TAG<1Ln-TAG. Oxidation stability of Lipase OF(®) (regiospecificity: random)-interesterified blended oils also improved on lowering the 3Ln-TAG content. In addition, the oxidation stabilities of Lipozyme RM-IM(®)-interesterified oils were slightly higher than those of the Lipase OF(®)-interesterified oils. We found that the content of 3Ln-TAG was almost the same in both oils, and the content of unsaturated fatty acid at the sn-1, 3 positions of Lipase OF(®)-interesterified oils was higher than that of Lipozyme RM-IM(®)-interesterified oils. These results indicate that oxidation stabilities of oils containing TAG with unsaturated fatty acid such as Ln at sn-2 position were higher than those having unsaturated fatty acids at the sn-1, 3 positions. From these results, the oxidation stability of oils rich in Ln, such as perilla oil and linseed oil, can be improved not only by decreasing 3Ln-TAG but also by enzymatically reducing the unsaturated fatty acid content at the sn-1, 3 positions.